The autophagy pathway is a critical component of antiviral innate immunity in which viruses are targeted for degradation inside lysosomes, but we know very little about how this is accomplished. We have performed the first unbiased screen for host factors involved in selective autophagy of viruses (virophagy), and identified 141 putative virophagy factors. We confirmed that one of these genes, the E3 ubiquitin (Ub) ligase and p62- interacting protein, Smurf1, is a novel virophagy factor for Sindbis virus (SIN) and herpes simplex virus (HSV)-1. We found that Smurf interacts with SIN capsid and that its role in selective autophagy of another substrate (mitochondria) is independent of its Ub ligase function. Aim 1 tests our hypothesis that Smurf1 targets p62- bound SIN and HSV-1 nucleocapsids for virophagy in a Ub ligase-independent manner. First, we will determine whether Smurf1's Ub ligase activity is required for virophagy by performing complementation experiments in SIN-infected Smurf1-/- MEFs, and we will use p62-/- MEFs to determine whether Smurf1:SIN capsid interaction requires p62. Then, we will determine whether Smurf1 also interacts with HSV-1 capsid, and whether its function in virophagy of HSV-1 is similar to that for SIN. Finally, we will characterize Smurf1:SIN and Smurf1:HSV-1 capsid complexes to identify additional host factors involved in Smurf1-mediated virophagy. Three other hits in the screen (FANCC, FANCF, and FANCL) are members of the Fanconi anemia (FA) core complex that assembles in response to DNA damage, one of which (FANCC) has a mouse model. FANCC has additional, DNA repair-independent, cytoprotective functions that overlap with functions of autophagy, are critical in the pathogenesis of FA, and are poorly understood. Aim 2 will confirm our preliminary data that FANCC plays a novel role in virophagy, explore the relationship FANCC's virophagy and cytoprotective functions, and determine if the FA core complex assembles in response to viral infection. First, we will assess the impact of FANCC deficiency on virophagy using FANCC-/- MEFs and fibroblasts from FANCC patients infected with SIN and HSV-1. Next, we will test whether FANCC mutants that dissociate its DNA repair and cytoprotective functions can also be segregated based on their role in virophagy, and, if so, determine whether these mutants differentially interact with SIN capsid and/or other host factors. Finally, we will test whether SI or HSV-1 infection triggers assembly and enzymatic activity of the complete FA core complex. Aim 3 will assess the role of Smurf1 and FANCC in host antiviral defense in vivo. First, we will confirm that targeting of viral nucleocapsids to autophagosomes is defective during SIN or HSV-1 CNS infection in Smurf1-/- or FANCC-/- mice. We will then determine whether Smurf1-/- or FANCC-/- mice are more susceptible to lethal CNS infections with SIN and HSV-1 and, if so, examine infected tissue to define the impact of defects in virophagy on viral pathogenesis. The studies outlined in this grant will give us new insight into the mechanism of virophagy and provide the first data regarding its importance in host defense against viruses in vivo. The goal of the research proposed in this grant is to deepen our knowledge of how cells use the autophagy pathway to eliminate invading viruses. We performed the first systematic search for human genes that regulate the targeting of viruses for destruction by autophagy, and we have preliminary data implicating several new genes in this pathway. Our study represents an important step toward a more detailed understanding of the function of autophagy in protection from viral infections, but further studies are necessary so we can ultimately harness its potential for treating viral diseases.